Humpback whales, like other polar wildlife, accumulate persistent organic pollutants. In Southern hemisphere populations, hexachlorobenzene (HCB) dominates the contaminant profiles. HCB is linked to a variety of health effects and is classified as a group 2B carcinogen, but the mechanism of action is a matter of contention. Potential toxicological effects to humpback whales remain entirely unknown. The recently established humpback whale fibroblast cell line (HuWa) offers an in vitro model for toxicological investigations. We here combine this novel cell line with a passive dosing strategy to investigate whale-specific toxicity of HCB. The relevant partitioning coefficients were determined to produce stable and predictable exposure concentrations in small-scale bioassays. The system was used to assess acute toxicity as well as genotoxicity of HCB to the HuWa cell line. While we found some transient reductions in metabolic activity, measured with the indicator dye alamarBlue, no clear acute toxic effects were discernible. Yet, a significant increase in DNA damage, detected in the alkaline comet assay, was found in HuWa cells exposed to 10 mu g L-1 HCB during the sensitive phase of cell attachment. Collectively, this work provides a ready-to-use passive dosing system and delivers evidence that HCB elicits genotoxicity in humpback whale cells.